title: Relaxin protects rat lungs from ischemia-reperfusion injury via inducible NO synthase: role of ERK-1/2, PI3K, and forkhead transcription factor FKHRL1.
abstract: BACKGROUND
  Early allograft dysfunction following lung transplantation is mainly an ischemia/reperfusion (IR) injury. We showed that relaxin-2 (relaxin) exerts a protective effect in lung IR, attributable to decreases in endothelin-1 (ET-1) production, leukocyte recruitment, and free radical generation. Here, we summarize our investigations into relaxin's signalling.
  
  
  METHODS
  Isolated rat lungs were perfused with vehicle or 5 nM relaxin (n = 6-10 each). Thereafter, experiments were conducted in the presence of relaxin plus vehicle, the protein kinase A inhibitors H-89 and KT-5720, the NO synthase (NOS) inhibitor L-NAME, the iNOS inhibitor 1400W, the nNOS inhibitor SMTC, the extracellular signal-regulated kinase-1/2 (ERK-1/2) inhibitor PD-98059, the phosphatidylinositol-3 kinase (PI3K) inhibitor wortmannin, the endothelin type-B (ETB) antagonist A-192621, or the glucocorticoid receptor (GR) antagonist RU-486. After 90 min ischemia and 90 min reperfusion we determined wet-to-dry (W/D) weight ratio, mean pulmonary arterial pressure (MPAP), vascular release of ET-1, neutrophil elastase (NE), myeloperoxidase (MPO), and malondialdehyde (MDA). Primary rat pulmonary vascular cells were similarly treated.
  
  
  RESULTS
  IR lungs displayed significantly elevated W/D ratios, MPAP, as well as ET-1, NE, MDA, and MPO. In the presence of relaxin, all of these parameters were markedly improved. This protective effect was completely abolished by L-NAME, 1400W, PD-98059, and wortmannin whereas neither PKA and nNOS inhibition nor ETB and GR antagonism were effective. Analysis of NOS gene expression and activity revealed that the relaxin-induced early and moderate iNOS stimulation is ERK-1/2-dependent and counter-balanced by PI3K. Relaxin-PI3K-related phosphorylation of a forkhead transcription factor, FKHRL1, paralleled this regulation. In pulmonary endothelial and smooth muscle cells, FKHRL1 was essential to relaxin-PI3K signalling towards iNOS.
  
  
  CONCLUSIONS
  In this short-time experimental setting, relaxin protects against IR-induced lung injury via early and moderate iNOS induction, dependent on balanced ERK-1/2 and PI3K-FKHRL1 stimulation. These findings render relaxin a candidate drug for lung preservation.
authors.affiliation: University Heart Center Dresden, Department of Cardiac Surgery, Dresden, Germany.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Alexiou
authors.forename: Manuel
authors.initials: M
authors.lastname: Wilbring
authors.forename: Klaus
authors.initials: K
authors.lastname: Matschke
authors.forename: Thomas
authors.initials: T
authors.lastname: Dschietzig
mesh_terms: D000818:Animals; D001794:Blood Pressure / Q000187:drug effects; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D019272:Leukocyte Elastase / Q000378:metabolism; D008168:Lung / Q000187:drug effects* / Q000473:pathology; D020935:MAP Kinase Signaling System / Q000502:physiology; D008315:Malondialdehyde / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D009195:Peroxidase / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011651:Pulmonary Artery / Q000187:drug effects; D051381:Rats; D012065:Relaxin / Q000378:metabolism / Q000494:pharmacology*; D015427:Reperfusion Injury / Q000517:prevention & control*; D014655:Vascular Resistance / Q000187:drug effects
chemical_list: D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D019272:Leukocyte Elastase; D008315:Malondialdehyde; D019001:Nitric Oxide Synthase; D009195:Peroxidase; D019869:Phosphatidylinositol 3-Kinases; D012065:Relaxin; C499515:FOXO3 protein, rat